Supported lipid bilayers have been used as a tool to study the biophysical properties of model membranes with defined compositions. Special attention has been given to the role of cholesterol on the phase behavior of lipid membranes, in particular, to the formation of lipid "rafts" or domains and complexes. Common techniques used to elucidate the phase behavior of binary and ternary lipid bilayers (i.e. fluorescence and atomic force microscopy) have been limited by their inability to provide direct information on the spatial composition of membranes. Secondary ion mass spectrometry (NanoSIMS) is a powerful tool for imaging the lateral organization and composition of lipid bilayers. Because the components in a lipid bilayer are uniquely identified by isotopic labels, NanoSIMS can unambiguously identify different molecules co-localized in a region as small as 50nm. In these studies, a method for producing cholesterol isotopomers at high levels of 13C enrichment is described. Furthermore, NanoSIMS will be used to image the spatial distribution and co-localization of cholesterol and proteins in native cell membrane fragments. PUBLIC HEALTH RELEVANCE: The lipid bilayer of a cell membrane is a complex and dynamic system in terms of lateral composition and organization. Living cells depend on the concerted rearrangement of membrane components, such as cholesterol, lipids, and proteins;yet very little is known about how this organization changes during essential processes, such as, cell-cell interactions, signal transduction, membrane trafficking, and membrane-bound protein activity and accessibility. In these studies, secondary ion mass spectrometry (nanoSIMS) imaging will be used to obtain quantitative compositional information with nanometer scale resolution of model and actual cell membranes.